Project Summary/Abstract Oxidative stress is an important mechanism of developmental toxicity, teratogenesis, and disease from environmental exposures. Developing animals rely on tightly regulated redox status for proper cellular differentiation and proliferation and thus are especially sensitive to oxidative stress. Susceptibility to oxidants is determined by both constitutive and inducible expression of antioxidant defenses, but how these are regulated during development is not well understood. In adults, the oxidative stress response is controlled in part by NF- E2-related factor 2 (NRF2) and related CNC-bZIP family proteins (NRF1, NRF3), which regulate transcription of genes involved in antioxidant defense, but the relative roles of NRF1, NRF2, and NRF3 in protecting developing animals from oxidative stress are largely unknown. The proposed basic research will investigate fundamental mechanisms underlying the response to oxidative stress during development using embryos of the zebrafish (Danio rerio), a well-established in vivo vertebrate model for studying developmental processes and chemical effects in developing animals. The overall objective of the research proposed here is to elucidate the mechanisms by which vertebrate embryos respond to oxidative stress during development, and how these responses are controlled by multiple NRF proteins. The central hypothesis is that NRF1, NRF2, and NRF3 have important but distinct roles in controlling antioxidant gene expression and the chemical-specific response to oxidants during development. The proposed studies will take advantage of several important features of the zebrafish embryo model, including evolutionarily conserved developmental processes, accessible embryos, the presence of gene duplicates (nrf1a/nrf1b, nrf2a/nrf2b), and the ability to rapidly perform gene targeting using CRISPR-Cas9 to generate loss-of-function and epitope-tagged alleles for elucidating gene function. Experiments to test the hypothesis that zebrafish nrf paralogs have undergone subfunction partitioning will yield new understanding about the roles of these proteins in development. Aim 1 will establish null or functionally compromised mutants of each nrf gene and thereby determine the roles of each Nrf protein in normal development and developmental gene expression (RNA-seq). Aim 2 will determine the role of each Nrf protein in embryotoxicity and the transcriptional response to structurally distinct model oxidants during development. In vivo promoter occupancy (embryo ChIP-exo-seq) and in vivo promoter analysis will identify genes that are targeted directly by Nrf proteins. Aim 3 will determine the role of each Nrf protein in the effects of diverse developmental toxicants. This basic research is the first comprehensive analysis of Nrf function in a developing vertebrate in vivo. The results will provide a foundation for hazard assessment, chemical screening, and additional mechanistic studies and provide insight into the role of Nrf proteins in protecting against oxidative stress in developmental disease.